PCR problems. I need to know what is going on…..

[micky_74]

So that´s the problem. I´m doing a specific PCR on a mutated sequence. The sequence is different from the wild type for 4 consecutives nucleotides. So one of my primer has the 3´sequence with the last four nucleotides on the different one (like an ARMS PCR). I´m working on a mosaicism (as not every cell of my culture is presenting the mutation)
I´m working on 96 wells plates. F1 cells taken from mouse. For extraction I treat the cells with lysis buffer plus 200 ug ml PK at 55 C for 30 minutes, then adding 1 volume of isopropanol precipitate DNA, then 1 wash with ET-OH 70%. Taking care that every well is dry, resuspend the pellet in water.
The PCR seems to work on a plasmid I made to optimize it. When I´m doing a plate (100 samples),but then when I repeat the experiment with the positives ones (30 samples usually) I got a smear. Some of them yesterday showed a clear band and also the plasmid diluted 1 to 10-6 showed a smear. The Tm of the primers is 60 C, I´m working at 58 with 2.5 mM mgcl2. I´m performing 45 cycles but going down to 40 is not helping at all.
Is the smear due to common primer as the sequence is present in wild type (the majority of them) and in the mutated? Is maybe the common primer creating the smear? Is the DNA which I assume is not very clean? A…I almost forgot……the smear is actually not going trough all the lane down, but I can see a brighter smear where the band should be (around 130 bp)
I ran a TD yesterday, I checked two different pairs of primers yesterday, but the result was the same…..a smear!

Anyone can come out with any suggestion?

[oppox]

I cant answer to the preparation steps before the PCR, one thing u can try though which is pretty fast. Do an extra heating to 95 deg after the PCR program and then cool it down. You have a pretty low working temp though and maybe it causes some problem.

what polymerase do u use?
Do u have both wiltype and mutated template present?
u have the "mutated" 3′ primer and what other primers present?
Have u tried and purify the PCR fragment using pcr purification kit of some kind?

[LilKim]

Hmmm…. I’ve never extracted DNA from mammalian cells like that… I’ve used trizol (or a kit) and both have worked really well. If I were to guess, i would guess that some of the insoluble debris left over in the pellet (after precipitation) is screwing up your PCR.

Because, that’s one of the biggest variables in you Plasmid only prep. Vs. lysed cell prep.

Well smearing could caused by….

1. Too much starting DNA (check by spec… check your purity and quality too!)
2. Poor quality DNA ( Your DNA might be getting degraded by DNAase’s BEFORE your proteinase K treatment)
3. Incorrect MgCl concentration.
4. Low annealing temperatures.

Also, whenever I precipitate DNA i’ve done it with 100% ethanol for at least 1 hour (in the freezer) then washed with 70% ethanol and then let it dry COMPELETLY (which can take some time if you don’t pull all of the etoh rightaway).

Also, i usually run 30 -35 cycles. (For whatever reason my boss swears by 30 … so that’s what I usually do)

okay that’s my 2 cents!

buena suerte and good luck!
– KIM

[micky_74]

Trying to answer both of you
Yes all the sequences are present (wild type and mutant- more wild type than mutant since lot of cells, the feeder ones, are wild type) I´m using only one pair of primers per reaction. Why should I clean up the PCR product? The band is there is it is there…….I´m not seeing the reason to purify the PCR product is I obtain one. It can be caused by too much DNA yes, but I specified that the smear is not covering the entire lane and I can not see the clear band at the interface comb-gel. I´m usign 1.5 mM Mgcl which in my opinion is quite low and 58C it is not too much low annealing temperature. From a 96 well plate how do you extract DNA by trizol? You have t work on each well taking ccare of taking the acqueous part? Pls let me know
Thank you for your ideas!

[LilKim]

Hey there !

hmmm .. Do see the desired band..IN your smear? Or is the desired band a huge smear? Or is there a smear of a certain size?

I was imagining that the smearing you saw was simlar to scenario 2 or 3… but i’d have to see the gel. (you can upload it on here if you want!)

Ya know what? Trizol extraction on 96 wells would be rough!! It would eat up a big chunk of your day. I think you may want to look into a commercial kit that isolates clean genomic DNA from mammalian cells. I’m sure that quiagen makes on … although i too would have to look for it.

Low MgCL2 would be less likely to increase non-specific amplification of your plasmid. However, I don’t exactly know what effect the PK prepped cells would have on MgCl concnetraionbecause we’re not sure if there is something else in your reaction that’s messing it up. (chelators??? … who knows?)

…in my experience when working with not-clean prepped DNA (for whole cell PCR of yeast) i have had to add ADDITIONAL MgCl2 to make the reaction work… and once your found the correct concentraion the reaction was quite robust.

Start of by getting a good OD to determine concentration and quality of your gDNA. That’s a good .. starting point to troubleshoot the rest.

good luck … let us know
– KIM

[canalon]

I definitely suspect your DNA extraction procedure to be the cause of your problems.

A good way to check would be to extract properly with trizol one of your experimental samples and see if you have PCR product in this condition. If yes, follow LilKim advice: Qiagen makes DNA purification kit adapted for 96 wells format (spin or vacuum) but they are expensive. If you have 30 sample at a time you can go with individual spin columns (from any manufacturer) without too much problem. It will take you less than a morning work (my experience is working with bacteria) and give you a DNA of much better quality than your extraction procedure.

[oppox]

hmm yea I re-read my answer and yea purifying it didnt make any sence. I picked up a gene from a genomic DNA were the primers needed had a annealing temp of 63 deg, I used two pairs of primers, the first ones had extra GC in the ends to raise the annealing temp and in the next run I used the primers with the low annealing temp without extra GC but matching the gene.
But I think u should try to lower your cycles to around 25-30 and see if it do anything. Maybe also lower the annealing temp to 55 and your extension temp.
I use 55 annealing and 68 deg in the extension step, using Taq polymerase.

Good luck.

[canalon]

The number of PCR cycles are not going to change much, since in fact after about 30/35 cycles the Taq polynerase is almost degraded.

Annealing temp depends of the primer, doing a gradient PCR could help you check if your smear is due to lack of specificity. And since you use a plasmid as a positive control, you may miss non-specific hibridization. You can also improve specificity by increasing MgCl2 concentration and/or adding DMSO to your reaction.

[LilKim]

additionally.. if you’re still having PCR problems after you get pure, clean DNA please let me know. When i’ve had problems in the past i’ve added Betaine to my reaction … and it was a beautiful thing.

So, please let me know if you’re still having problems… I can look up the optinamal betaine Vs. DNA concentration to make things work …. HOPEFULLY

– KIM

[micky_74]

Actually
I found out the problem: contamination. I knew 90% of the times when you have a smear is due to some sort of contamination……..so my dummy has the same smear
I will try to change primers

I will let you know!!

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